Anthrax is a disease is believed to be caused by the spore-forming bacterium, Bacillus anthracis, a bacterium that is readily found in soil. B. anthracis is believed to primarily cause disease in plant-eating animals. Though infrequent, when humans do become infected, they usually acquire the bacterium from contact with infected animals, animal hides or hair, or animal feces. The human disease has a relatively short incubation period (less than a week) and usually progresses rapidly to a fatal outcome.
In humans, anthrax may occur in three different forms: coetaneous anthrax, gastrointestinal anthrax and inhalation anthrax. Coetaneous anthrax, the most common form in humans, is usually acquired when the bacterium, or spores of the bacterium, enter the body through an abrasion or cut on the skin. The bacteria multiply at the site of the abrasion, cause a local edema, and a series of skin lesions—papule, vesicle, pustule and necrotic ulcer—are sequentially produced. Lymph nodes nearby the site are eventually infected by the bacteria and, in cases where the organisms then enter the bloodstream (20% of cases), the disease is often fatal. Gastrointestinal anthrax is caused by eating contaminated meat. Initial symptoms include nausea, vomiting and fever. Later, infected individuals present with abdominal pain, severe diarrhea and vomiting of blood. This type of anthrax is fatal in 25% to 60% of cases. Inhalation anthrax (also called woolsorters' disease) is acquired through inhalation of the bacteria or spores. Initial symptoms are similar to those of a common cold. Symptoms then worsen and these individuals present with high fever, chest pain and breathing problems. The infection normally progresses systemically and produces a hemorrhagic pathology. Inhalation anthrax is fatal in almost 100% of cases.
Coetaneous anthrax is acquired via injured skin or membranes, entry sites where the spore germinate into vegetative cells. Proliferation of vegetative cells results in gelatinous edema. Alternatively, inhalation of the spores results in high fever and chest pain. Both types may be fatal unless the invasive aspect of the infection may be intercepted.
B. anthracis is believed to possess two major virulence components. The first virulence component is a polysaccharide capsule which contains poly-D-glutamate polypeptide. The poly-D-glutamate capsule is not itself toxic but plays an important role in protecting the bacterium against anti-bacterial components of serum and phagocytic engulfment. As the B. anthracis bacterium multiplies in the host, it produces a secreted toxin which is the second virulence component of the organism. This anthrax toxin mediates symptoms of the disease in humans.
The anthrax toxin is believed to comprise three distinct proteins encoded by the bacterium: protective antigen (PA), lethal factor (LF) and edema factor (EF). PA is the component of the anthrax toxin that is believed to bind to host cells using an unidentified cell-surface receptor. Once it binds to cell surfaces, EF or LF may subsequently interact with the bound PA. The complexes are then internalized by the host cell with significant effects. EF is an adenylate cyclase which causes deregulation of cellular physiology, resulting in edema. LF is a metalloprotease that cleaves specific signal transduction molecules within the cell (MAP kinase isoforms), causing deregulation of said pathways, and cell death. Injection of PA, LF or EF alone, or LF in combination with EF, into experimental animals produces no effects. However, injection of PA plus EF produces edema. Injection of PA plus LF is lethal, as is injection of PA plus EF plus LF.
As an acute, febrile disease of virtually all warm-blooded animals, including man, anthrax can be used in biological weapons (BW). For example, ten grams of anthrax spore may kill as many people as a ton of the chemical warfare agent, sarin. Terrorists have included dry spores in letters. Biological weapons of mass destruction have been developed that contain large quantities of anthrax spores for release over enemy territory. Once released, spores may contaminate a wide geographical area, infecting nearly all susceptible mammals. Due to the spore's resistance to heat and dry conditions, contaminated land may remain a danger for years. In view of the serious threat posed by the disease, effective diagnostic tools are needed to assist in prevention and control of natural and man-made outbreaks. Due to the highly lethal nature of anthrax and BW agents in general, there is great need for the development of sensitive and rapid BW agent detection. Current detection technology for biological warfare agents have traditionally relied on time-consuming laboratory analysis or onset of illness among people exposed to the BW agent.
One promising approach to the detection and treatment of B. anthracis is the use of bacteriophage lysins as bacteriolytic agents. Bacteriophages specific for B. anthracis and related B. cereus bacteria strains may be isolated and used to detect and treat these bacteria. Bacteriophages near B. anthracis spores during spore germination may be used to infect and lyse the bacteria. A variety of phage-based bacterial therapies have been reviewed. D. H. Duckworth, P. A. Gulig, “Bacteriophages: Potential treatment for bacterial infections,” BioDrugs, 16(1), 57-62 (2002). There are various environmental bacteriophages present in soils that may infect and lyse B. anthracis under controlled conditions. H. W. Ackermann, et al., “New Bacillus bacteriophage species,” Archives of virology, 135(3-4), 333-344 (1994); H. W. Ackerman, M. S. Dubrow, Viruses of prokaryotes: General properties of bacteriophages, Boca Raton, Fla., CRC Press, Inc. (1989);
A bacterial lysin called PlyG, from bacteriophage-γ of B. anthraci, has been shown to lyse vegetative B. anthracis cells and is useful in promising methods for treatment of anthrax. R. Schuch, D. Nelson, V. Fischetti, “A bacteriolytic agent that detects and kills Bacillus anthracis,” Nature 418, 884-889 (2002), incorporated herein by reference. A nucleotide sequence encoding PlyG is disclosed in GenBank accession #AF536823 and has a molecular mass of about 27,000. PlyG has been shown to control anthrax disease in mice, and to bind to vegetative cells. However, PlyG has no means to replicate itself in the presence of host bacteria. Methods and composition for the treatment of a variety of bacterial infections using a phage associated lytic enzyme specific for the invasive bacteria and an appropriate carrier for delivering the lytic enzyme into a patient are discussed in the following U.S. patents issued to Fischetti et al.: U.S. Pat. Nos. 5,604,109; 5,985,271; 6,056,954; 6,056,955 6,248,324; 6,254,866; and 6,264,945, all incorporated herein by reference. Effective treatment of 14 of 24 virulent B. anthracis strains by phage based methods has been reported in a preliminary study done at Johns Hopkins University Applied Physics Laboratory. Michael Walter, Ph.D., “Efficacy and Durability of Bacilus anthracis Bacteriophages Used Against Spores,” Journal of Environmental Health, July/August 2003, 9-15.
Bacteriophages for B. anthracis may be isolated from the environment. For instance, Walter et al. report the isolation of Phages Nk, DB and MH for B. anthracis in topsoil. Walter, M H, Baker, D D, “Three Bacillus anthracis bacteriophages from topsoil,” Curr Microbiol. 2003 July; 47(1): 55-58. Further bacteriophages useful for detection and treatment of B. anthracis are reported herein. The W and γ environmental bacteriophages of B. anthracis have been identified in topsoil, but the isolation of the polynucleotide and the identification of open reading frames coding for various polypeptides therein were unknown. E. W. McCloy, “Studies of a lysogenic Bacillus strain. I. A bacteriophage specific for Bacillus anthracis,” Journal of Hygiene, 49(1), 114-125 (1951); E. R. Brown, W. B. Cherry, “Specific identification of Bacillus anthracis by means of a variant bacteriophage,” Journal of Infectious Diseases, 96(1), 34-39 (1955).
The direct introduction of bacteriophages into an animal to prevent or fight diseases has certain drawbacks. Specifically, both the bacteria and the phage have to be in the correct and synchronized growth cycles for the phage to attach. Additionally, there are preferably the right number of phages to attach to the bacteria; if there are too many or too few phages, there will be either no attachment or no production of the lysing enzyme. The phage is preferably active enough to be effective. The phages may also be inhibited by many things including bacterial debris from the organism it is going to attack. Further complicating the direct use of a bacteriophage to treat bacterial infections is the possibility of immunological reactions within the subject being treated, potentially rendering the phage non-functional. The ability of bacteriophages to lyse and kill target bacterial may also be decreased by sunlight, UV light, desiccation or other conditions encountered during storage or use of a phage-containing therapeutic agent. Therefore, the potential effectiveness of any given bacteriophage against a target bacteria depends on the conditions under which the phage is deployed against the target bacteria. Studying the structure of phages and their efficacy against target bacteria in various conditions are useful in developing therapeutic methods for treating and preventing disease caused by target bacteria. Investigations of the structure and function of phages may also relate to diagnostic methods for detecting target bacteria and spores, such as those of B. anthracis. Many environmental conditions that may alter the effectiveness of a phage, such as phage W and phage-γ, against a B. anthracis or related target bacteria. The isolation and analysis of the phage polynucleotide sequences, and associated polypeptide sequences, of these and other phages are needed to relate to effective methods for prevention, treatment and diagnosis of B. anthracis bacteria and spores.